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WO1988005309A1 - Augmentation de la puissance de conjugues cytotoxiques - Google Patents

Augmentation de la puissance de conjugues cytotoxiques Download PDF

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Publication number
WO1988005309A1
WO1988005309A1 PCT/US1988/000243 US8800243W WO8805309A1 WO 1988005309 A1 WO1988005309 A1 WO 1988005309A1 US 8800243 W US8800243 W US 8800243W WO 8805309 A1 WO8805309 A1 WO 8805309A1
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antigen
immunoglobulin
immunoglobulins
bind
epitope
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PCT/US1988/000243
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English (en)
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Vera S. Byers
Robert W. Baldwin
Patrick J. Scannon
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Xoma Corporation
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Publication of WO1988005309A1 publication Critical patent/WO1988005309A1/fr
Priority to KR1019880701179A priority Critical patent/KR890700357A/ko

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to cancer therapy and, more particularly, to the potentiation or enhancement of the cytotoxic properties of immunotoxins by the co-administration of a second, unconjugated monoclonal antibody.
  • Colorectal cancer is the second most common cause of death from malignancy in the Western world.
  • the American Cancer Society estimates that ther were 138,000 new cases of colorectal cancer and 59,900 patients died from the disease in 1985.
  • the outlook for patients with colorectal disease has remained essentially unaltered over the last 30 years, with the five- year survi val being on the order of 30%.
  • a major factor contributing to this is the lack of effective treatment for the disease once it has spread beyond the bowel wall, since surgery during the early stages of the disease offers the only prospect of cure.
  • Ultrasound, laparoscopy or peritoneoscopy, and CAT scan are of limited value in the diagnosis of ovarian carcinoma.
  • Serum markers such as carcinoembryonic antigen and placental alkaline phosphatase as well as some newly defined antigens are found in the blood of some patients with adenocarcinoma, although there is no universal marker.
  • Surgery with biopsy is the only definitive way of diagnosing ovarian carcinoma.
  • Radioisotope implants, x-ray irradiation, and chemotherapy are of limited use in the management of ovarian carcinoma .
  • Stage I ovarian carcinoma (growth limited to the ovaries) has an overall 5-year survival rate of approximately 80%
  • Stage II growth involving ovaries with pelvic extension
  • Stage III growth involving ovaries with extension to small bowel or omentum
  • Stage IV disant metastases
  • the relative 5-year survival rate for ovarian carcinoma is 37% (1973-80), relatively the same as for 1960-63 (32%). Imaging studies of ovarian carcinoma with radiolabelled MoAbs have been performed to a limited degree in human and animal systems.
  • Limb salvage procedures have been performed, such as en-block resection and prosthetic replacement. Overall survival for limb salvage is either as poor or worse than with amputation. Radiation treatment has not been shown to prevent OS metastases.
  • the described invention relates to potentiating the cytotoxicity of antibodies conjugated to a toxin, ie., an immuunotoxin.
  • a toxin ie., an immuunotoxin.
  • the enhancement of cytotoxicity is demonstrated by a cytotoxic cocktail containing an immunotoxin to which is added a second immunoglobulin that binds to a second epitope on the same antigen to which the immunotoxin binds.
  • an immunotoxin consisting of a first immunoglobulin conjugated to a toxin, said first immunoglobulin directed against a cell surface antigen, preferably a tumor associated antigen
  • the method comprises exposing a target cell expressing the antigen with the immunotoxin and a second immunoglobulin in a dose stifficient to potentiate said immunotoxin, said second immunoglobulin able to bind to a different epitope on the antigen than that. bound by said first immunoglobulin.
  • the first and second immunoglobu l ins are able to bind to an epitope on a carcinogenicembryonic antigen.
  • the epitope bound by the second immunoglobulin is within the normal cross-reacting antigen subsection of the carcinogenicembryonic antigen.
  • Immunoglobulins binding to T-cells both normal and leukemic are useful in the disclosed method. Particularly useful are immunoglobulins to the CD-5 antigen.
  • Specific and preferred monoclonal antibodies for use in this invention and their hybridomas are also described herein.
  • a cytotoxic therapeutic cocktail comprising: a conjugate including a toxin bound to a first immunoglobulin, said first immunoglobulin able to bind to an epitope on a cell surface antigen, preferably a tumor associated antigen; and a second immunoglobulin in a dose sufficient to potentiate said immunotoxin, said second immunoglobulin able to bind to a different epitope on the antigen than that bound by said first immunoglobulin. It is preferred that the cytotoxic therapeutic cocktail embrace first and second immunoglobulins that are able to bind to an epitope on the carcinogenicembryonic antigen.
  • the second immunoglobulin binds to an epitope that is located within the normal cross-reacting antigen subsection of the carcinogenicembryonic antigen.
  • Cocktails containing immunoglobulins binding to T-cells both normal and leukemic are also useful in this invention. Particularly useful are immunoglobulins to the CD-5 antigen. It should be noted that it is not necessary to physically combine the immunotoxin and second immunoglobulin. Separate administration would also constitute a cocktail as defined herein.
  • an immunotoxin which consists of a first immunoglobulin conjugated to a toxin said immunoglobulin directed against a tumor associated antigen
  • the method comprises exposing cancer cells expressing the antigen to the immunotoxin and to a second immunoglobulin in a dose sufficient to potentiate said toxin, said second immunoglobulin able to bind to a different epitope on the antigen than that bound by said first immunoglobulin.
  • the first and second immunoglobulins are able to bind to an epitope carcinogenicembryonic antigen.
  • the epitope bound by the second immunoglobulin is within the normal cross-reacting antigen subsection of the carcinocembryonic antigen.
  • This method is useful against cancer cells selected from the group consisting of colorectal carcinoma cells, gastric cancer cells, pancreatic cancer cells, lung cancer cells, and breast cancer cells.
  • Immunoglobulins binding to T-cells both normal and leukemic are useful in the disclosed method.
  • Particularly useful are immunoglobulins to the CD-5 antigen.
  • Preferred dosages are approximately 0.01 mg to 20.0 mg per kg of host body weight per day and most preferably the doses are approximately 0.05 mg to 5.0 mg per kg of host body weight per day.
  • the preferred ratio of said second immunoglobulin to said immunotoxin is 10,000 - 0.001 to 1 by weight and most preferably the ratio of second immunoglobulin to said immunotoxin is 100 - 0.01 to 1 by weight.
  • a second epitope it is meant that the two antibodies bind to physically different locations on their target antigen. It is preferred that the binding not be competitive but a slight amount of competition due to close proximity is acceptable. Potentiation can be quantified and the effect of competition may be offset by appropriate changes in the rati os of immunotoxin to immunoglobulin. Monoclonal antibodies are preferred for use in this invention.
  • Figure 1 is a graphic illustration of the influence of free RTA and XMMCO-228-RTA on the growth of colorectal carcinoma LS174T cell xenografts.
  • the present invention employs cytotoxic conjugates for the treatment of various forms of cancer, including colorectal carcinoma, ovarian carcinoma and osteogenic sarcoma.
  • novel conjugates are comprised of monoclonal antibodies or binding fragments thereof, collectively termed immunoglobulins, bound to a cytotoxin.
  • These compositions are admini stered to a cancer cell host in order to destroy cancer cells whi le doing minimal damage to normal tissue.
  • the immunoglobulins of the present invention are employed as targeting agents for directing cytotoxic agents to specific cancer cells within a cancer cell host.
  • immunoglobulins which define an epitope on a 72 kilodalton (kD) glycoprotein antigen and immunoglobulins which define an epitope on carcinoembryonic antigen (CEA) are employed as targeting agents.
  • kD 72 kilodalton
  • CEA carcinoembryonic antigen
  • One or both of these antigens are present on a variety of cancers including, but not limited to, colorectal carcinoma, ovarian carcinoma and osteogenic sarcoma.
  • cytotoxic agents are suitable for use in immunotoxins.
  • the cytotoxic agents contemplated by this invention can include radionuclides. such as Iodine-131, Yttrium-90, Rhenium-188, and Bismuth212; a number of chemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, and cisplatinum; and cytotoxic proteins such as ribosomal inactivating proteins including, pokeweed antiviral protein, abrin and ricin (or their A-chains), diphtheria toxin pseudomonas exotoxin A or recombinant derivitives. etc. See generally, "Chimeric Toxins", Olsnee and Phil.
  • Toxic lectins are of particular interest in this invention.
  • the cytotoxic action of toxic lectins, and especially that of ricin and abrin, has been well studied. It is known that toxic lectins consist of two polypeptide chains, A and B, linked by means of disulfide bridge (s). Cytotoxicity is associated with the A chain and its inhibition of protein synthesis in nucleated cells.
  • the B chain is essentially a delivery vehicle for the A chain.
  • the B chain recognizes polysaccharide units at the surface of cells and creates a high affinity interaction with such units. Once the B chain binds with polysaccharide units at the cell surface, the A chain is incorporated into the cell, blocking ribosomal protein synthesis and ultimately leading to cell death.
  • the use of ricin A chain is preferred in this invention and its use is as described in U.S. Patent No. 4,590,071, the disclosure of which are hereby incorporated by reference.
  • a preferred form of ricin toxin A chain for use in this invention is one wherein substantially pure RTA-30 is used.
  • RTA-30 refers to a species of ricin toxin A chain having a molecular weight of approximately 30 kD, such as described in detail by Fulton et al. J. Biol. Chem., 281:5314-5319 (1986) and Vidal et al. Int. J. Cancer, 36:705-711 (1985).
  • RTA preparations containing concentrations of about 75% or more of RTA-30 are considered substantially pure. Preparation of substantially pure RTA-30 for use in conjunction with a MoAb is described in U.S. Patent Application Serial Number 074 , 824 which is incorporated herein by reference.
  • the immunoglobulin and toxin are generally bound by a covalent bond, more particularly a disulfide bond, but may be joined by any chemical bond which allows the toxin to travel to the target cell with the immunoglobulin.
  • a covalent bond more particularly a disulfide bond
  • Other methods for achieving such a bond are well-known to those skilled in the art. The only criteria is that the bond must be achieved in a manner which does not significantly decrease the binding affinity of the immunoglobulin for its epitope.
  • the present cytotoxic conjugates may be administered to a cancer cell host either singly or in a cocktail containing two or more conjugate formulations, other chemotherapeutic agents, compositions, or the like. Cocktails are particularly important in the treatment of heterogeneous tumor cell populations wherein targeting of multiple antigens is critical.
  • the cytotoxic conjugates of the present invention may be administered to a cancer cell host by any convenient method.
  • Pharmaceutical compositions employing the subject conjugates may be administered parenterally, i.e., intravenously, intraparitoneally, or the like.
  • this invention provides compositions for parenteral administration which comprise a solution of pyrogen free, cytotoxic conjugates, or a cocktail thereof, dissolved in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, or the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well-known filtration sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjus ting and buffering agents, toxicity adjusting agents, or the like, for example, sodium acetate, sodium chloride, potassium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • the compositions of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use.
  • lyophilization and reconstitution can lead to varying degrees of antibody activity loss and that use levels may have to be adjusted to compensate.
  • Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • the pharmaceutical formulation should provide a quantity of conjugate (s) of this invention sufficient to effectively treat the cancer cell host.
  • the dose of conjugate or conjugate cocktail will vary widely, generally from about 0.01 mg/kg/day to 20.0 mg/kg/day, usually about 0.05 mg/kg/day to 10.0 mg/kg/day, and, more particularly, 0.05 mg/kg/day to 5.0 mg/kg/day.
  • the dose of unconjugated immunoglobulin will vary widely depending upon the immunotoxi n employed, choice of unconjugated immunoglobulin, nature and extent of the tumor, and the like.
  • the dose will generally be in the range of 0.001 mg/kg/day to 100.00 mg/kg/day, usually about 0.01 mg/kg/day to 10.0 mg/kg/day and, more particularly, about 0.1 to 1 mg/kg/day.
  • the ratio of unconjugated immunoglobulin to conjugate will also vary widely, generally from about 10,000-0.001 to 1, usually about: 1000-0.01 to 1 and, more particularly, about 100-0.1 to 1.
  • Kits may also be supplied for use with the subject conjugates or cocktails.
  • the subject compositions may be provided, usually in lyophilized form, either alone or in conjunction with additional chemotherapeutic agents for cancer therapy.
  • These kits may include buffers, such as phosphate buffered saline, other inert ingredients, or the like.
  • the kits may also include specific instructions including suggested protocols for the administration of the subject compositions to a cancer cell host.
  • the efficacy of the subject immunotoxin compositions may be potentiated by the co-administration, via a similar route, of an unconjugated immunoglobulin in conjunction with the subject immunotoxins. Potentiation means that the efficacy of the immunotoxin, in terms of target cell kill, is increased for a given dose of immunotoxin.
  • mice (Bantin & Kingman, U.K.) were immunized with carcinoembryonic antigen (CEA) derived from a perchlorate extract of a colon carcinoma liver metastasis.
  • CEA carcinoembryonic antigen
  • the immunization schedule consisted of 10 ⁇ g CEA in complete Freund's adjuvant (CFA) given intraperitoneally on Days 0 and 7, and 20 ug of CEA (Also in CFA) given intraperitoneally on Days 25, 56 and 63.
  • CFA complete Freund's adjuvant
  • spleen cells from an immunized mouse were aseptically removed. Following procedures as outlined elsewhere (Galfre et al (1977) Nature 266:550, which is incorporated by reference), 10 6 spleen cells were fused with 10 6 cells of P3-NSI-Ag4-1 (A.T.C.C. Accession No. TIB-18), a hypoxanthine-methotrexate-thymidine (HMT) sensitive murine myeloma cell line. Using polyethylene glycol (PEG), hybrid cells were placed into 96-well culture plates,
  • Fetal Calf Serum Myoclone, Gibco, Paisley, U.K.
  • hypoxanthine 10 -4 M
  • thymidine 1.6 x 10 -5 M
  • methotrexate 10 -5 M
  • EIA enzyme immunoassay
  • XMMCO-228 The clone designated XMMCO-228 was found to stably secrete immunoglobulin which was determined to be of the IgG2a subclass by solid phase RIA using standard methods.
  • Hybridoma XMMCO-228 is presently on deposit with the American Type Culture Collection (A.T.C.C), 12301 Parklawn Dr., Rockville, MD 20852, USA. The deposit was made on August 14, 1986, and given A.T.C.C. Accession No. HB 9174.
  • mice Balb/c mice (Bantin & Kingman, U.K.), 6-10 weeks old, were used to culture the hybridoma peritoneally. Approximately 10 7 hybridoma cells were injected into mice that had been pretreated 3 weeks earlier with 0.5 mls of pristane (Aldridge, Gillingham, Dorset.
  • the antibody in ascites fluid was purified by affinity chromatography using a Sepharose - Protein A column using methods well known by those skilled in the art.
  • the hybridoma was grown in vitro in Dulbecco s
  • the binding of XMMCO-228 to MKN45 cells was determined by flow cytometry employing methods well known to those skilled in the art and described above.
  • the gastric carcinoma cell line MKN45 expresses both the 72kD antigen with which the XMMCO-791 MoAb reacts and the CEA antigen with which XMMCO-228 reacts.
  • B14 is a MoAb directed against breast cancer, which reacts with the normal cross-reacting antigen (NCA) subsection of the CEA antigen. It is described in Example II. Normal mouse serum was used as a control. The results are summarized in Table I.
  • XMMCO-228 was evaluated for crossreactivity with normal tissues using the indirect immunoperoxidase conjugate technique.
  • tissues were simultaneously tested with a purified murine MoAb in the same concentration.
  • the control immunoglobulins used for these purposes were an IgG2 mouse myeloma protein (UPC 10, Litton Bionetics, Kensington, MD) or a MoAb with reactivity to sheep red blood cells (SRBC).
  • OCT Optimal Cutting Temperature Compound
  • the specimen was "snap" frozen in liquid nitrogen-cooled i sopentane ( 2-methylbutane , VWR, Norwald , CA) at controlled temperature (-120 to -150°C) for 10-15 seconds.
  • the ti ssue was kept frozen for long-term storage at -70°C, or for short-term storage at -20°C, in a tightly sealed container to prevent evaporation and drying of tissues.
  • Frozen ti ssues were allowed to equilibrate slowly overnight by placing in a -35°C cryostat prior to cutting the frozen sections. Cut sections were dried overnight at room temperature prior to staining.
  • a circle was etched on each slide around the tissue using a diamond pencil. Slides were then fixed in acetone for 1 minute, followed by a 10 minute PBS wash. Excess fluid was removed and the primary antibody applied for one hour while making sure that the tissue was covered and not allowed to dry out, followed by a 10 minute PBS wash. Excess fluid was removed and peroxidase-con- jugated goat anti-mouse IgG (Tago/product code 6450) (diluted 1:10 in PBS) applied for 30 minutes, followed by a 10 minute PBS wash.
  • the slides were developed in aminoethylcarbazole (AEC), (0.5 ml AEC stock 120 mg AEC in 15 ml dimethylformamide), 9.5 ml acetate buffer (79 ml of 0.1 M sodium acetate, 21 ml of 0.1 N acetic acid), and 0.05 ml 3% H 2 O 2 ) for 5 minutes, followed by a 5 minute H 2 O wash.
  • AEC aminoethylcarbazole
  • the slides were then counterstained with fresh Mayer s Hemtoxylin for 5 minutes, followed by a 5 minute wash in running tap water and covers lipped with glycerine mounting media.
  • Table II is a summary of the results:
  • NCA Normal colon antigen
  • D. XMMCO-228 Ricin Toxin A Chain ( RTA ) Conjugates The conjugation technique, including purification of the A chain of ricin, is disclosed in U.S. Patent No. 4,590,071, the disclosures of which are hereby incorporated by reference.
  • mice 7-10 nude mice were implanted with LS174T cells, a human colon adenocarcinoma derived cell line (A.T.C.C. Accession No. CL 188).
  • LS174T cells a human colon adenocarcinoma derived cell line
  • CL 188 a human colon adenocarcinoma derived cell line
  • a cocktai l of XMMCO-791-RTA (Scand. J. Immuno. 18:411-420 (1983) and XMMCO-228-RTA consists of a mixture of XMMCO-791-RTA and XMMCO-228-RTA. in any ratio, and administered to a cancer cell host in a dose range of about 0.01 mg/kg/day to 20.0 mg/kg/day.
  • the cocktail is administered parenterally, generally by intravenous infusion or intrapercitoneal injection in a suitable vehicle, such as phosphate buffered saline or the like.
  • Balb/c mice were immunized with 521AM whole cells derived from ascites breast metastasis.
  • the immunization schedule consisted of 6 intraperitoneal injections of 10 6 cells at weekly intervals.
  • a final intravenous boost of 3x10 5 cells was given 3 days prior to fusion.
  • spleen cells from an immunized mouse were aseptically removed.
  • a single cell suspension was obtained using an 80 mesh wire screen grid #1985-00080 (Bellco). Cells were washed with Iscoves Complete Medium (Grand Island. N.Y., N.Y.) and counted.
  • Sp2/0-Ag14 cells A.T.C.C. Accession No. CRL 1581
  • HAT hypoxanthine-aminopterin-thymidine
  • the two cell types were fused at a ratio of 2 spleen cells per myeloma cell.
  • the fusion products were plated into 96-well culture plates at a concentration of 10 5 myeloma cells per well.
  • Cells were cultured in Iscoves with 20% fetal bovine serum and ⁇ mercaptoethanol with HAT medium (hypoxanthine 136 mg/100 ml, aminopterin .018 mg/100 ml, thymidine 136 mg/100 ml).
  • Hybridoma XMMBR-B14 is presently on deposit with the American Type Culture Collection (A.T.C.C), 12301 Parklawn Dr., Rockville, MD 20852, USA. The deposit was made on January 14, 1987, and given A.T.C.C. Accession No. HB 9308.
  • Balb/c mice (Bantin & Kingman, U.K.), 6-10 weeks old, were used to culture the hybridoma peritoneally. Approximately 10 7 hybridoma cells were injected into mice that had been pretreated 3 weeks earlier with 0.5 mis of pristane (Aldridge, Gillingham, Dorset, U.K.) injected intraperitoneally (i.p.).
  • the antibody in ascites fluid was purified by affinity chromatography using a Sepharose Protein A column using methods well known by those skilled in the art.
  • the hybridoma was grown and cloned in vitro in Iscoves Medium with 20% fetal bovine serum and ⁇ mercaptoethanol in plastic 300 ml bottles.
  • Cell concentration was 10 5 cells/ml medium over a culturing period of 4-5 days, with a MoAb concentration of 4 ⁇ g/ml medium, and a doubling time of 12 hours.
  • XMMBR-B14 The binding of XMMBR-B14 to cell lines and primary carcinoma-derived cells was determined by flow cytometry employing methods well known by those skilled in the art and described above. The tests (Table VI) showed that XMMBR-B14 detects an antigen expressed on breast and colon carcinoma cell lines. Further studies using purified protein preparations have established that the epitope defined by XMMBR-B14 is found on an antigen of the classification termed CEA/NCA (carcinoembryonic antigen/normal cross-reacting antigen). This is the portion of the CEA molecule that also cross-reacts with exposed normal cross-reacting antigen. It also reacts with cells derived from two primary colon carcinomas. Normal mouse immunoglobulin (NMIg) or normal mouse serum (NMS) were used as controls.
  • NMIg normal mouse immunoglobulin
  • NMS normal mouse serum
  • XMMBR-B14 was tested for reactivity with primary colon carcinoma membrane and normal colonic mucosa using standard EIA methods well known by those skilled in the art.
  • the test summarized in Table VII, shows that XMMBR-B14 reacts with colon carcinoma membrane.
  • TP 1 - membrane preparation from pooled primary colon carcinoma TP 1 - membrane preparation from pooled primary colon carcinoma.
  • NP 1 - membrane preparation from pooled normal colonic mucosa from colon cancer patient.
  • T186 membrane preparation - primary colon carcinoma T186.
  • XMMBR-B14 The Reactivity of XMMBR-B14 with CEA and NCA preparations was determined by a solid phase radioimmunoassay. Briefly, antigen preparations were coated into wells of microtest plates. Monoclonal antibody was then added, incubated 1 to 2 hours and wells washed.
  • Table IX shows the increase in cytotoxicity of XMMCO-228-RTA immunotoxin against MNK45 cells when increasing amounts of XMMBR-B14 are added.
  • the present invention provides efficacious, novel compounds and methods for the therapy of various cancer cells.
  • the cytotoxic conj ugates are particularly efficacious when administered in cocktai l form or in the presence of certain unconjugated monoclonal antibodies.

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Abstract

La présente invention se rapporte en général à la thérapie anticancer et, plus particulièrement, à l'augmentation de la puissance ou à l'amélioration des propriétés cytotoxiques d'immunotoxines par l'administration combinée d'un deuxième anticorps monoclonal non conjugué.
PCT/US1988/000243 1987-01-27 1988-01-26 Augmentation de la puissance de conjugues cytotoxiques WO1988005309A1 (fr)

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US704587A 1987-01-27 1987-01-27
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PCT/US1988/000243 WO1988005309A1 (fr) 1987-01-27 1988-01-26 Augmentation de la puissance de conjugues cytotoxiques

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EP (1) EP0299057A4 (fr)
JP (1) JPH01502195A (fr)
KR (1) KR890700357A (fr)
WO (1) WO1988005309A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0337746A2 (fr) * 1988-04-12 1989-10-18 City Of Hope National Medical Center Composition pour augmenter la biodistribution d'anticorps pour localisation dans des lésions
EP0419462A1 (fr) * 1987-07-17 1991-04-03 Xoma Corporation Therapies ameliorees a base d'immunotoxines utilisant des especes a chaine-a de ricine purifiee
EP0489931A1 (fr) * 1990-06-29 1992-06-17 Toray Industries, Inc. Complexe immunotoxinique
US5173293A (en) * 1989-02-23 1992-12-22 Becton Dickinson And Company Anti-T-cell antibodies as adjuvants
US6184043B1 (en) 1992-09-14 2001-02-06 FODSTAD øYSTEIN Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations
US6265229B1 (en) 1994-03-10 2001-07-24 Oystein Fodstad Method and device for detection of specific target cells in specialized or mixed cell populations and solutions containing mixed cell populations

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US4590071A (en) * 1984-09-25 1986-05-20 Xoma Corporation Human melanoma specific immunotoxins
EP0098162B1 (fr) * 1982-06-30 1991-05-15 Yuji Matsuoka Anticorps monoclonaux spécifiques de l'antigène carcinoembryonnaire

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EP0098162B1 (fr) * 1982-06-30 1991-05-15 Yuji Matsuoka Anticorps monoclonaux spécifiques de l'antigène carcinoembryonnaire
US4590071A (en) * 1984-09-25 1986-05-20 Xoma Corporation Human melanoma specific immunotoxins

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Title
CHEMICAL ABSTRACTS, Volume 102, No. 15, 15 April 1985, (Colombus, Ohio, USA), GRIFFIN et al, "Enhancement of the Specific Cytotoxicity of Anti-Carcinoembryonic Carboxylic Ionophores," Abstract No. 130074n, Protides Biol. Fluids, (1984), 32, 449-53. *
CHEMICAL ABSTRACTS, Volume 104, No. 19, 12 May 1986, (Colombus, Ohio, USA), CASELLAS et al, "Potentiation of Cytotoxicity Induced by Immunotoxins", Abstract Number 161626q, Ucla. Symp. Mol. Cell. Biol., New Ser., 1985, 27, 263-74. *
Proceedings of the National Academy of Sciences, Volume 77, August 1980 (USA) "Antibody-Directed Cytotoxic Agents: Use of Monoclonal Antibody to Direct the Action of Toxin A Chains to Colorectal Carcinoma Cells", see entire document. *
See also references of EP0299057A4 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0419462A1 (fr) * 1987-07-17 1991-04-03 Xoma Corporation Therapies ameliorees a base d'immunotoxines utilisant des especes a chaine-a de ricine purifiee
EP0419462A4 (en) * 1987-07-17 1991-07-17 Xoma Corporation Improved immunotoxin therapies utilizing purified ricin a-chain species
EP0337746A2 (fr) * 1988-04-12 1989-10-18 City Of Hope National Medical Center Composition pour augmenter la biodistribution d'anticorps pour localisation dans des lésions
EP0337746A3 (fr) * 1988-04-12 1991-10-09 City Of Hope National Medical Center Composition pour augmenter la biodistribution d'anticorps pour localisation dans des lésions
US5173293A (en) * 1989-02-23 1992-12-22 Becton Dickinson And Company Anti-T-cell antibodies as adjuvants
EP0489931A1 (fr) * 1990-06-29 1992-06-17 Toray Industries, Inc. Complexe immunotoxinique
EP0489931A4 (en) * 1990-06-29 1993-02-24 Toray Industries, Inc. Immunotoxin complex
US6184043B1 (en) 1992-09-14 2001-02-06 FODSTAD øYSTEIN Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations
US6893881B1 (en) 1992-09-14 2005-05-17 Abbott Laboratories, Inc. Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations
USRE43979E1 (en) 1992-09-14 2013-02-05 Abbott Laboratories Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations
US6265229B1 (en) 1994-03-10 2001-07-24 Oystein Fodstad Method and device for detection of specific target cells in specialized or mixed cell populations and solutions containing mixed cell populations

Also Published As

Publication number Publication date
EP0299057A1 (fr) 1989-01-18
EP0299057A4 (fr) 1990-02-22
JPH01502195A (ja) 1989-08-03
KR890700357A (ko) 1989-04-24

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